Type 1 diabetes mellitus (T1DM) arises through autoimmune destruction of pancreatic cells and results in life-long dependency on daily insulin injection. Epidemiological data show that T1DM incidence is rising by 2-3% per year while onset is occurring at an earlier age. Since acquisition of relevant immune tissues from prediabetic children is nearly impossible, we have used congenic derivatives of the BioBreeding (BB) rat. The lymphopenic DRIyp/lyp animal develops spontaneous T1DM, whereas the DR+/+ does not. Evidence points to disease progression in BB rats being dependent on the absence of natural regulatory T (nTreg) cells. T1DM in BB rats also involves cytotoxic T cells, since their depletion is protective. Our preliminary studies show that BB rats are predisposed to T1 DM in that pancreatic cells of both DRIyp/lyp and DR+/+ strains recruit immune cells through expression of eotaxin, an eosinophil and mast cell recruiting chemokine, as well as interleukin 1 prior to insulitis in the DRIyp/lyp rat. Gene expression studies of prediabetic pancreatic lymph nodes (PLN) have shown that mast cells are terminally activated in DRIyp/lyp animals versus.negatively regulated in DR+/+ animals. We have successfully delayed T1DM in DRIyp/lyp rats with two different mast cell inhibiting drugs, confirming a pathogenic role for mast cells. Thus, we propose that nTreg cells in the DR+/+ rat can maintain the islet health by a peripheral regulatory mechanism, whereas in the absence of nTreg cells, DRIyp/lyp animals spontaneously progress to T1DM. We hypothesize that in the BB rat, innate immune cells mediate islet destruction unless nTreg cells prevent their mobilization. We will test our hypotheses through the following aims: 1) Temporally and spatially dissect the activities of nTreg cells during the prediabetic period at day 30 (prior to islet eotaxin signaling), day 40 (prior to insulitis), day 50 (early insulitis) and day 60 (late insulitis, pre-onset) using a combination of functional genomics and histological approaches. 2) Validate Aim 1 observations through in vitro and in vivo studies that characterize the action of nTreg on CD8 and CD4 T cells as well as innate cells. A role for mast cells in initiating and/or amplifying autoimmunity in T1DM has only recently been defined. These studies will utilize genomic, bioinformatic, histological, and immunological approaches to elucidate the relationship between innate cells and nTreg cells. This integrated approach will help us better understand how mast cells and nTreg cells interact and has the potential to establish a new paradigm for immune regulation, thus opening new insights into immune disease.